Recent research on human pluripotent stem cells such as human ES cells (hESCs) and human iPS cells (hiPSCs) provides an increased potential for practical applications of regenerative medicine. Since such cells have an unlimited proliferative capacity and a differentiation capability into various cells, regenerative medicine using pluripotent stem cells is expected to fundamentally change methods for treatment of refractory diseases, lifestyle-related diseases, and other diseases. Recent techniques have already enabled the induction of differentiation of pluripotent stem cells in vitro into various cells, including nerve cells, cardiac muscle cells, blood cells, and retinal cells.
Human pluripotent stem cells, such as hESC and hiPSC, have been conventionally cultured primarily on feeder cell layers using mouse embryonic fibroblasts (MEFs). Feeder cells supply growth factors that are useful for maintenance culture of human pluripotent stem cells to stem cells. The activity to enable maintenance culture of human pluripotent stem cells has been found in various human cell types, in addition to MEFs (Hovatta O. et al., Hum. Reprod., (2003) 18, pp. 1404-1409; Richards M. et al., Nat. Biotechnol., (2002) 20, pp. 933-936; Cheng L. et al., Stem Cells, (2003) 21, pp. 131-142; and Richards M. et al., Stem Cells, (2003) 21, pp. 546-556). However, in conventional techniques, the preparation of feeder cells at the time of culture has been laborious, and there is a risk of contamination of feeder cells in stem cells. Therefore, the development of alternative safer techniques has been needed.
As a pluripotent stem cell culture technique without using MEFs, a method involving the use of a MEF-preliminarily conditioned medium (i.e., MEF-CM) from a medium supplemented with a serum such as FBS or a serum replacement and a method with the use of chemically fixed MEFs (Yue X-S. et al., PLoS. ONE, (2012) 7, e32707) are known. In addition, a method involving the use of various human cells (e.g., fibroblasts, placental cells, bone marrow cells, and endometrial cells) as living feeder cells without using xenogeneic cells has been reported (Hayato Fukusumi and Yonehiro Kanemura, Development of Feeder-free Cell Culture Technique of Human ES/iPS Cells, Journal of Clinical and Experimental Medicine, (2011) 239, pp. 1338-1344). In these methods, a medium supplemented with bovine serum or KNOCKOUT™ SR (Knockout Serum Replacement, which is an additive used as a serum replacement for enabling culture of ES/iPS cells) is used in order to culture human pluripotent stem cells. However, many of the additive components comprise proteins extracted from bovine serum. Accordingly, infectious diseases such as bovine spongiform encephalopathy (BSE) and viral contamination of cells become issues of concern. While human serum is used on some occasions, human serum is not suitable for practical use because of restrictions in use thereof or quantitative limitations.
In addition, the development of complete synthetic medium (chemically defined medium) for culture without MEFs has been advanced (Akopian V. et al., In Vitro Cell Dev. Biol. Anim., (2010) 46, pp. 247-258; and Chen G. et al., Nat. Methods, (2011) 8, pp. 424-429). MEF secretory products are analyzed for functional proteins (Chin A. C. et al., J. Biotechnol., (2007) 130, pp. 320-328). However, it remains difficult to stably culture human pluripotent stem cells in feeder-free, serum-free culture, and development of a technique that enables satisfactory growth has been awaited.